Method for quickly screening taxol-producing endophytic fungi

A technology of endophytic fungi and paclitaxel, which is applied in biochemical equipment and methods, and microbial measurement/inspection, etc., can solve the problems of high cost, insufficient accuracy, time-consuming consumables, etc., and achieve a breakthrough in time-consuming and labor-intensive screening. process, the effect of improving screening efficiency

Inactive Publication Date: 2009-04-29
HUAZHONG UNIV OF SCI & TECH
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Problems solved by technology

However, although thin-layer chromatography is simple and cheap, the detection accuracy is not enough; although high-performance liquid chromatography, mass spectrometry, monoclonal antibody immunoassay and other methods are sensitive and accurate, their cost is also high
If dozens or hundreds of fungi are mass-cultured, paclitaxel is extracted and purified, and these sensitive and accurate detection methods such as high performance liquid chromatography, mass spectrometry, and monoclonal antibody immunoassay are used for detection and screening, it will be a very important task. time consuming work

Method used

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  • Method for quickly screening taxol-producing endophytic fungi

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Effect test

Embodiment 1

[0041] The trunk bark of Taxus media cv. Hicksii was collected from the campus nursery of Huazhong University of Science and Technology, and cut into small pieces (~0.3×0.5×0.3cm). Small pieces are processed and the endophyte is isolated. The small pieces of the bark inner epidermis were placed on the PDA medium plate containing 50 μg / mL ampicillin, and cultured in an incubator at 23 °C. 167 endophytic fungi.

[0042] 167 strains of endophytic fungi were respectively inoculated into 30 mL of potato dextrose liquid medium, cultured at 23°C and 120 rpm for 3 days, and the genomic DNA of these fungi was extracted. There are three groups in C, and another group D, which contains 0.2 μL of genomic DNA of 67 fungal strains.

[0043] The PCR reaction system consisted of mixed fungal genomic DNA for one screening group, 50 μM dbat-F and 50 μM dbat-R, 100 μM dNTPs, 5 μL 10×PCR reaction buffer, 1 units Taq DNA polymerase, sterilized ddH 2 O make up to 50 μL.

[0044] PCR amplificati...

Embodiment 2

[0052] The stem bark tissue of Taxus yunnanensis Cheng et L.K.Fu was collected from Wuhan Botanical Garden, cut into small pieces (~0.5×0.5×0.5cm) with a sterile knife, and then isolated by conventional endophytic fungi isolation method. Small pieces of tissue were processed and endophytic fungi were isolated. Small pieces of the inner epidermis of the bark were placed on a PDA medium plate containing 75 μg / mL ampicillin, and cultured in an incubator at 26 °C. The endophytic fungi were purified, and finally 31 endophytic fungi were obtained.

[0053] 31 strains of endophytic fungi were respectively inoculated into 30 mL of potato dextrose liquid medium, cultivated at 26° C. and 150 rpm for 3 days, and the genomic DNA of these fungi was extracted, and 0.3 μL of each was taken for group screening. Each 10 strains of fungal genomic DNA was a screening group. There are two groups A and B, and another C screening group containing 11 strains of fungal genomic DNA. The dbat gene wa...

Embodiment 3

[0062] The near-root bark of Taxus cuspidata Sieb.et Zucc. was collected from the Yoga Mountain on the campus of Huazhong University of Science and Technology, cut into small pieces (~0.1×0.3×0.5cm) with a sterile knife, and then the conventional endophytic Isolation of fungi The tissue pieces are processed and endophytic fungi are isolated. The small pieces of the inner epidermis of the bark were placed on a PDA medium plate containing 100 μg / mL ampicillin, and cultured in an incubator at 28 °C. 45 endophytic fungi.

[0063] 45 strains of endophytic fungi were respectively inoculated into 50 mL of potato glucose liquid medium, cultured at 28° C. and 200 rpm for 5 days, the genomic DNAs of these fungi were extracted, and 0.5 μL of each was taken for group screening. Each 5 strains of fungal genomic DNA was a screening group. There are 9 groups from A to I. The dbat gene was amplified by using the mixed fungal genomic DNA as a template.

[0064] The PCR reaction system consis...

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Abstract

The invention discloses a method for quickly screening taxol-producing endogenetic fungi. The method quickly screens the taxol-producing endogenetic fungi by adoption of key enzyme genes synthesized by taxol as molecular markers and on the basis of the PCR amplification technology. The method designs primers according to conserved regions of 10-Deacetyl Baccatin III-10-O- acetylase genes and C-13phenylalanine side chain CoA acetylase genes of reported yew, performs amplification screening on fungi which is obtained by separation of yew barks in turn, performs fermentation culture on the fungi which can generate the two genes by means of amplification, extracts the taxol, analyzes the taxol of the fungi by HPLC and MS, and finally determines the taxol-producing endogenetic fungi. The invention provides an effective strain separating and screening method and a batch of effective original strains in order to realize replacement of extraction and conversion of the taxol from yew plant tissue by the microorganism fermentation method.

Description

technical field [0001] The invention belongs to the fields of microbial biotechnology and biopharmaceuticals, in particular to a method for rapidly screening taxol-producing endophyte fungi. Background technique [0002] Paclitaxel is an important antitumor drug recognized internationally. Due to the lack of taxus resources, the price of paclitaxel is expensive, which affects its wide clinical application. The use of microbial fermentation to produce paclitaxel is one of the effective ways to solve the problem of paclitaxel sources. [0003] Since Stierle et al. first reported the isolation of a taxol-producing endophytic fungus from the Pacific yew tree in 1993, only dozens of taxol-producing endophytic fungi have been reported at home and abroad. One of the reasons is that taxol-producing endophytic fungi screening is not easy. Up to now, the screening method for taxol-producing endophytic fungi is still similar to the method used by Stierle et al. The extract was anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 张鹏余龙江周蓬蓬付春华赵春芳
Owner HUAZHONG UNIV OF SCI & TECH
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